CaMKIIδ is required for targeted recycling of the LBRC. (A) iHUVECs grown on collagen gels and transduced with dominant negative (DN) CaMKIIδ as in Fig. 4 were subjected to the targeted recycling assay (described in the Materials and methods). This assay follows the recruitment of the LBRC to the site of transmigration. Using immunofluorescence microscopy, recycled LBRC is visualized as increased fluorescence adjacent to transmigrating monocytes. The arrow in the control panel highlights LBRC recruitment adjacent to the transmigrating monocyte while the arrow in the dominant negative panel highlights the notable absence of LBRC recruitment. Images shown here are representative of three independent experiments. Scale bar is 10 µm. (B) Quantification of the monocyte position and LBRC recruitment shown in A. Monocytes were considered at the junction if they overlapped at all with the endothelial cell (EC) junctions in the x-y plane. Data shown here are the average and standard deviation of three independent experiments. Each experiment had at least 50 monocytes scored. Data from each experiment were normalized to the total adherent cells to account for subtle differences in adhesion on each monolayer. ** denotes P value < 0.01 with Student’s t test.